Plants which are substantially “tolerant” to a herbicide when they are subjected to it provide a dose/response curve which is shifted to the right when compared with that provided by similarly subjected non tolerant like plants. Such dose/response curves have “dose” plotted on the x-axis and “percentage kill”, “herbicidal effect” etc. plotted on the y-axis. Tolerant plants will typically require at least twice as much herbicide as non tolerant like plants in order to produce a given herbicidal effect. Plants which are substantially “resistant” to the herbicide exhibit few, if any, necrotic, lytic, chlorotic or other lesions when subjected to the herbicide at concentrations and rates which are typically employed by the agricultural community to kill weeds in the field.
Within the context of the present invention the terms hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (HPPD), 4-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (4-HPPD) and p-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (p-OHPP) are synonymous.
Methods for providing plants which are tolerant to HPPD herbicides which comprise transformation of plant material with polynucleotides comprising regions which encode HPPD enzymes are known. However what has not hitherto been generally recognized is that different HPPD enzymes provide different levels of tolerance to different HPPD-inhibitor herbicides. While a given HPPD enzyme may provide a useful level of tolerance to some HPPD-inhibitor herbicides it may be quite inadequate to provide commercial levels of tolerance to a different, more desirable HPPD-inhibitor herbicide which, for example, may control a different spectrum of weeds, be cheaper to make or offer environmental benefits. As well as particular HPPD enzymes and the polynucleotides which encode them the current invention also provides a means of selecting HPPD enzymes suitable for providing commercially useful levels of resistance to particular HPPD-inhibitor herbicide chemistries.
In order to provide for plants with tolerance to commercially useful application rates of a desired HPPD herbicide it would be an advantage to use polynucleotides which encode HPPD enzymes having reduced susceptibility to inhibition by the desired HPPD herbicide or class of HPPD herbicides. This characteristic of reduced susceptibility to inhibition by HPPD herbicides in vitro is also expressed herein as ‘increased resistance’ or ‘inherent tolerance’.
Some mutant forms of a Pseudomonas sp. HPPD are claimed to exhibit such increased resistance on the basis of exhibiting an apparently decreased rate of binding of inhibitor to the enzyme (i.e., on the basis of measurements essentially corresponding to kon in the equilibrium E+I←→EI, vide infra). However such mutant enzyme forms have reduced catalytic activity and/or reduced stability which, potentially, renders them unsuitable for use especially in the warm climate crops, particularly corn and soybean for which HPPD-inhibitor herbicides generally provide the most useful spectrum of weed control. It has not hitherto been known that various unmutated wild-type HPPD enzymes from different sources can equally exhibit useful and different inherent levels of tolerance and that, furthermore, unmutated wild-type enzymes are preferable for use in transgenic plants because, in general, they exhibit considerably better stability and activity (kcat/Km) than the mutant derivatives.
Furthermore it has not hitherto been appreciated that the level of inherent tolerance of these wild-type HPPD enzymes or indeed of mutated HPPD enzymes can vary markedly according to the particular class and structure of HPPD inhibitor. Neither has it been known that these differences in tolerance have their basis not in differences in the parameter kon, addressed by previously used assay methods, but rather, in the parameters Kd, and koff. It has also not been appreciated that these differences in inherent tolerance can be marked and useful even between HPPD enzymes having relatively similar amino acid sequences as, for example, between sequence similar HPPD enzymes derived from different species of plants. In order to maintain the widest range of options for herbicide modes of action useful for the control of volunteer crops and to minimize any potential impact of gene flow to weeds it is desirable that the herbicide tolerance conferred upon transgenic plants be expressed preferentially toward only certain desired subclasses of HPPD inhibitor herbicides. This is another benefit of being able to choose a particular HPPD enzyme most suited to delivering resistance to a particular set of HPPD herbicide types.